The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Semen analysis is the most commonly used procedure to evaluate male fertility potential. This study was to evaluate the quality of 10 JBC bulls belonging to Jeju Special Self-Governing Province Promotion Institute. [JBC A∼J grade]. The freezing medium (20% egg yolk plus 20% triladyl) was added in semen sample to a final concentration of 100×106 sperm/ml. For sperm cooling, diluted semen was filled in 0.5 ml plastic straws and then kept in refrigerator at 4°C for 2 h. They were placed in 7 cm over liquid nitrogen (LN2) vapor for 10 min and then directly plunged into LN2 for storage. Thawing was done by transferring the frozen straws into water bath at 37°C for 30 sec for analysis. The sperm motility, vitality and morphology in each group was assessed using the Sperm Analysis Imaging System (SAIS Plus; Medical Supply Co, Ltd., Korea), eosin-nigrosin stain and diff-quik kit. There was no difference in the motility of the fresh groups (87.4 ~ 100%), while it was difference in the frozen-thawed groups (42.8 ~ 98.6%) (p<0.05). The best motility was shown in JBC-B (100/fresh and 98.6%/frozen-thawed). There was significant difference in the vitality of the fresh group (19.8 ~ 59.2%) and frozen-thawed group (21.2 ~ 49.8%)(p<0.05). The highest vitality was also shown in JBC-B (59.2/fresh and 49.8%/frozen-thawed). Morphologically, in fresh semen the highest normal ratio was indicated in JBC-E (90.9%) and in frozen-thawed group the highest was in JBC-C (90.2%). These results demonstrated that the analysis including motility, vitality and morphology of fresh or frozen-thawed semen is valuable to select the high quality sperm using for reproduction.

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

Sperm recovery from epididymis in animals considered as important tools to preserve high-value or endangered species. However, there are no appropriate castrating indicators of timing for recovery of sperm which can be available to artificial reproduction technologies such as artificial insemination (AI) and in vitro fertilization (IVF), particularly in young Hanwoo bull. Therefore, this study aimed to investigate semen volume, morphology and motility of sperm in epididymis of young Hanwoo bulls at 8, 13, 14, and 15 months of age. About 2 cm of the epididymal tail only was cut and minced using blades. Minced epididymal tail tissues were mixed with semen extender (OptixCell, France, IMV technologies) and sperm were recovered with a cell strainer (100 μm nylon mesh). The number of sperm at 8 months of age was lower than that at 13, 14, 15 months of age in bulls after collection (33.6±27.2 vs. 352.4±39.2, 320.4±113.6 and 422.8±252.4×107cells respectively; P<0.05). After the frozen-thawed sperm the the percentage of abnormal head, tail and dead damaged acrosome did not differ between the ages 13, 14 and 15 months of age in bulls (P>0.05), however, the dead sperm with intact acrosome (DIA), the numbers showed that more than 15 months in 8, 13, 14 months (8.7±4.1 vs. 47.3±12.2, 34.8±14.0, 28.8±8.5, P<0.05). In addition, frozen-thawed sperm at 8 months of age showed low total motile sperm compared to those at 13, 14 and 15 months of age (26.4±8.2 vs. 45.7±29.5, 62.3±41.0, 70.4±15.9%, respectively; P<0.05). In conclusion, sperm derived from epididymal tail at 8 months of age in Hanwoo bulls showed high abnormal morphology and poor motility, which is not adequate for artificial insemination(AI) and in vitro fertilization(IVF). On the other hand, sperm derived from epididymal tail at 13, 14, 15 months of age in bull showed high normal morphology and motility, which may be available for AI and IVF. Epididymal sperm collected from bulls over 13 months is needed for further study whether to use the actual in vitro fertilization.

For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Phospholipase C zeta (PLCz) is important enzyme in spermatogenesis, but their effect has not been confirmed in pigs yet. Therefore, this study was aimed to analyze their association with sperm motility and kinematic characteristics. DNA samples from 124 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity (VCL), Straight-line velocity (VSL), the ratio between VSL and VCL (LIN), Amplitude of Lateral Head displacement (ALH)] were subjected. A SNP in non-coding region of PLCz g.158 A > C was associated with MOT (p < 0.05), VCL (p < 0.01), LIN (p < 0.01) and ALH (p < 0.05) in Duroc population. Therefore, we suggest that the intron region of the porcine PLCz gene may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm–egg fusion, and was therefore selected as candidate gene for boar semen motility and kinematic characteristics. This study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 96 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.358A>T) in intron 6 was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine CD9 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts, and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm–egg fusion, and was therefore selected as a candidate gene to investigate Duroc boar semen motility and kinematic characteristics. This study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 96 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT, 82.27±5.58), Curvilinear velocity(VCL, 68.37±14.58), Straight-line velocity(VSL, 29.06±6.58), the ratio between VSL and VCL(LIN, 47.36±8.42), Amplitude of Lateral Head displacement(ALH, 2.88±0.70)] were used in present study. A single nucleotide polymorphism (g.358A>T) in intron 6 was associated with MOT, VCL, VAP and ALH in Duroc population (p<0.05). Therefore, we suggest that the porcine CD9 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts, and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

The recovery of epididymal sperm in animals is considered as one of the important tools to preserve high value or endangered species. However, there are no appropriate castrating indicators such as months of age in bull, sperm morphology, and motility, particularly in young Korean native bull (Hanwoo). Therefore, this study aimed to investigate sperm number, morphology, and motility of sperm in the epididymis tail of young Hanwoo bulls at 8 and 15 months of age. After castration, epididymal tails were collected and minced with blades to recover sperm. In experiments 1 and 2, sperm number, morphology, and motility were examined. Total number of sperm and percentage of normal sperm from bulls at 8 months of age was lower than that of bulls at 15 months of age after collection (P<0.05). Percentage of abnormal head, tail, proximal cytoplasmic droplet, dead and damaged acrosome of sperm from bulls at 8 months of age were higher than those of bulls at 15 months of age (P<0.05). In experiment 3, sperm motility from bulls at 8 and 15 months of age were examined before freezing and after thawing. Frozen-thawed sperm at 8 months of age showed low total motility and motile sperm with ≥ 25 μm/sec compared to those at 15 months of age and commercially-used sperm (P<0.05). In conclusion, sperm derived from the epididymal tail of bulls at 8 months of age showed high abnormal morphology and poor motility, which are not adequate for AI and IVF. On the other hand, sperm derived from the epididymal tail of bulls at 15 months of age showed high normal morphology and motility.

This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability (84±1.15 %, p<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability (79±3 %, p<0.05) than those of the X and Y sperm (44.7±1.67 and 41.7±1.2 %) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities (24.3±1.2 and 25.7±0.9 %, p<0.05) compared to that of the sorted Y sperm (13.7±1.2 %) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher (55.0±1.7 and 45.0±1.5 %) than those of the sorted Y-sperm (32.3±0.9 %, p<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

본 연구는 체세포를 이용하여 생산된 복제 한우 수소의 번식능력을 검토하기 위해 실시하였다. 복제 한우 수소(C-38 및 C-39) 또는 일반 한우 종모우로부터 정액을 채취하여 정자의 수 및 동결 전후의 생존성 등을 살펴보았으며, 정자의 운동성 등은 computer assisted sperm analysis(CASA)를 이용하여 측정하였다. 또한, 이들의 수정 능력을 확인하기 위하여 체외수정과 인공수정을 각각 실시하였다. 정액 성상에서는 복제 수소들과 일반 종모우 간에 정액의 양, 정자의 농도 및 동결융해 후의 생존성 등에서 차이가 나타나지 않았다. CASA를 이용한 분석에서 운동성, 곡선 운동 속도(VCL), 직선 운동 속도(VSL) 및 평균 진행 속도(VAP) 등은 복제 수소의 정액이 일반 종모우의 정액에 비하여 유의적으로 높았다(p<0.05). 체외수정에 따른 수정란의 분화율 및 배반포로의 발달율은 복제 수소와 일반 종모우 간에 차이가 나타나지 않았다. 복제소 정액(C-38)을 이용하여 인공수정을 한 5두의 체세포 복제 대리모에서 암수 각각 한 두씩의 건강한 복제 후대 송아지 2두를 생산하였다. 이상의 결과를 종합하여 보면, 실험에 공시된 복제 수소 개체 간의 차이가 나타나기는 하였지만, 복제 수소는 정액 성상과 정자의 운동성 등에서 일반 종모우와 차이가 없었으며. 또한 인공수정을 통해 송아지를 생산함으로써 정상적인 번식능력이 있음을 확인하였다.

본 연구는 제주도 축산진흥원과 농촌진흥청 난지농업연구소에서 사육되고 있는 제주마 5두를 사용하여 인공질을 이용, 정액을 채취하여 정액의 일반성상 및 CASA를 이용하여 정자의 운동성과 생사 염색으로 기형률을 조사하였다. 제주마 정액의 일반 특성으로 총 정액량은 평균 42.5ml이었으며 평균 pH는 7.3, 평균 정자농도는 평균 이었다. 정자의 운동성은 이며, motility parameters 별로 VAP 로 나타났다. 정자 부위별 기형률은 두부가 평균

This study was carried out to evaluate the effects of washing medium, breed and washing temperature of fresh and frozen-thawed boar sperm on mitochondrial activity and membrane integrity by flow cytometry. More than 80% of fresh sperm washed with mTLP-PVA medium at 20℃ exhibited an intact membrane and a functional mitochondrion. With frozen-thawed samples, a large number of sperm showed both damaged membrane (36.4~46.9%) and nonfunctional mitochondrion (55.1~71.1%) in the mTLP-PVA and BTS washing media at 20℃. There were no breed effects of fresh and frozen-thawed sperm on mitochondrial activity and membrane integrity. The percentages of damaged membrane of fresh and frozen sperm, respectively, were higher at 4℃ washing temperature than at 20℃ washing temperature in the mTLP-PVA medium. We found that washing medium and washing temperature of fresh and frozen-thawed boar sperm were important for the analyses of mitochondrial activity and membrane integrity by flow cytometry.

The purpose of this study was the analysis of sperm ability in Specific Pathogen Free (SPE) miniature pig for production of bio-organ. The collected semen was diluted with extender and stored at 17℃ for up to 7 days. The semen samples were evaluated at 0, 1, 3, 5, and 7 days of storage for analysis of sperm ability. Sperm ability was evaluated by examining viability, progressive motility, sperm abnormality and intensity of the sperm membrane. Also, the semen was processed according to the convenient freezing method, and frozen-thawed sperm was evaluated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) staining. Motility of spermatozoa of SPF miniature pig was significantly (P<0.05) lower on 3 days or later compared to the Duroc, Yorkshire and Landrace in domestic boar. The percentage of abnormal spermatozoa of Landrace were significantly (P<0.05) higher than in SPF miniature pig, Duroc and Yorkshire that had a similar percentage on 5 or 7 days of sperm storage. The percentage of spermatozoa with coiled tail decreased during the storage period but there were no significant difference. On the other hand, viability of frozen-thawed spermatozoa had a significantly (P<0.05) lower in SPF miniature pig than in other domestic boars. CTC patterns had no significant difference, but SPF miniature pig had higher percentage of capacitated spermatozoa and lower percentage of acrosome-reacted it than domestic boars. Therefore, this study suggest that it is necessary to develop the suitable extender and freezing methods methods for the high viable rate and fertilizing ability in vitro.

Among proteins separated from methanol extract of follicular fluid with superose column, the components inducing sperm swim-up separation through sucrose layer were analysed with superose column in Smart system and SDS-PAGE. And the results obtained were as follows; The fractions of retention volume (RV) 0.83ml and RV 1.36ml separated with superose column should stimulate sperm migration and movement. However, RV 0.83 fraction was consisted of complex materials containing RV 1.36 component. RV 1.36 fraction contained a BSA analogue of 67 kilodaltons (Kd) and showed identical peak pattern with BSA fraction V. In conclusion, the protein of 67 Kd in follicular fluid should stimulate sperm migration and movement.